Assessing genetic diversity in Ziziphus jujuba ‘Jinsixiaozao’ using morphological and microsatellite (SSR) markers

Ziziphus jujuba ‘Jinsixiaozao’ is one of the most elite Chinese jujube variety with a long cultivation history. There are many different types and newly developed accessions of ‘Jinsixiaozao’ reported, however the names are in chaotic. For the accurate identification of the accessions and estimating the genetic diversity, the diversity and relationships of the 45 ‘Jinsixiaozao’ accessions were evaluated by 49 morphological traits and 24 highly polymorphic genomic SSR primers. The UPGMA dendrogram based on morphological traits separated the accessions into four major groups with Euclidean distance ranging from 4.26 to 12.26. Six of 24 SSR primers produced polymorphic patterns with a total of 17 alleles. Cluster analysis using UPGMA and Jaccard‘s coefficient grouped the accessions into eight groups. The SSR markers failed to distinguish the majority of the analyzed accessions, and a negative percentage of variation was partitioned. These results indicated low genetic diversity among the collected accessions. The mantel test revealed a weak negative correlation (r = −0.051) between the morphological dissimilarity matrix and that based on SSR markers.     

Inter-primer binding site retrotransposon and inter-simple sequence repeat diversity among wild Lens species

Even though lentil has been an important food legume for centuries, genetic studies in lentil are still in their infancy. Genetic diversity and relationships among wild Lens species from Turkey has seldom been investigated. Additionally, a limited number of simple sequence repeat (SSR) markers have been developed for use in breeding and genetic studies of lentil crop. In this study, molecular characterization of 50 accessions mostly from Turkey, belonging to 6 wild and 1 cultivated Lens species, was performed using newly developed inter-primer binding site (iPBS) retrotransposons and inter-SSR (ISSR) markers. The 10 iPBS primers generated a total of 151 scorable bands, of which 150 were polymorphic (99.3%) with an average of 15.0 polymorphic fragments per primer. The 10 ISSR primers detected 138 scorable bands showing 100% polymorphism, with an average of 13.5 bands per primer. The average polymorphism information content (PIC) value for ISSR markers (0.97) was higher than that for iPBS markers (0.90). Lens orientalis was found to be the most diverse species, raising the possibility of wide crosses with cultivated species Lens culinaris. Cultivated varieties also showed high level of polymorphism, at 82.92% and 51.92% with ISSR and iPBS markers, respectively. Lens lamottei and Lens tomentosus were found as the least polymorphic species using both marker systems. The grouping of accessions and species within clusters were almost similar when iPBS and ISSR graphs were compared. Our data also suggested the role of iPBS-retrotransposons as ‘a universal marker’ for molecular characterization of wild and cultivated Lens species.     

Diversification of indigenous gene- pool by using exotic germplasm in lentil (Lens culinaris Medikus subsp. culinaris)

Genetic diversity was studied among 21 accessions of lentil using SSR markers and morphological traits in order to assess the diversification of Indian gene-pool of lentil through introgression of exotic genes and introduction of germplasm. Among these , 16 genotypes either had ‘Precoz’ gene, an Argentine line in their pedigree or genes from introduced lines from ICARDA. Sixty five SSR markers and eight phenotypic traits were used to analyse the level of genetic diversity in these genotypes. Forty three SSR markers (66 %) were polymorphic and generated a total of 177 alleles with an average of 4.1 alleles per SSR marker. Alleles per marker ranged from 2 to 6. The polymorphic information content ranged 0.33 to 0.80 with an average of 0.57, suggesting that SSR markers are highly polymorphic among the studied genotypes. Genetic dissimilarity based a dendrogram grouped these accessions into two main clusters (cluster I and cluster II) and it ranged 33 % to 71 %, suggesting high level of genetic diversity among the genotypes. First three components of PCA based morphological traits explained higher variance (95.6 %) compared to PCA components based on SSR markers (42.7 %) of total genetic variance. Thus, more diversity was observed for morphological traits and genotypes in each cluster and sub-cluster showed a range of variability for seed size, earliness, pods/plant and plant height. Molecular and phenotypic diversity analysis thus suggested that use of germplasm of exotic lines have diversified the genetic base of lentil germplasm in India. This diversified gene-pool will be very useful in the development of improved varieties of lentil in order to address the effect of climate change, to adapt in new cropping systems niches such as mixed cropping, relay cropping, etc. and to meet consumers’ preference.     

Microsatellite marker-based identification and genetic relationships of olive cultivars from the Extremadura region of Spain

Seventy-seven olive accessions corresponding to 25 cultivars from the Extremadura region of Spain were studied using four microsatellite or SSR markers in order to fingerprint them, and evaluate genetic similarity and relationships between local and introduced olive cultivars. The number of alleles per locus ranged from 4 to 8, with a mean of 6.25 alleles per primer pair (a total of 25 alleles). The observed heterozygosity ranged from 0.58 to 0.95, while the expected heterozygosity varied between 0.68 and 0.83. The polymorphism information content values ranged from 0.63 to 0.79. The mean polymorphism information content value of 0.70 for the SSR loci provided sufficient discriminating ability to evaluate the genetic diversity among the cultivars. The SSR data allowed unequivocal identification of all the cultivars; a combination of three SSR markers was sufficient to discriminate all 25 olive cultivars. A dendrogram was prepared, using the unweighted pair-group method with arithmetic mean clustering algorithm; it depicted the pattern of relationships between the cultivars. Most of the local cultivars grouped according to their geographic origin. No clear clustering trends were observed when the morphological traits of fruit endocarps or fruit use of cultivars were employed as analysis criteria. We conclude that there is a high level of variability among local olive cultivars from the Extremadura region at both the morphological and molecular levels; these data should be useful for identifying and distinguishing local germplasm.     

The use of molecular markers for germplasm management in a French olive collection

With more than 100 accessions, the CBNMP olive collection includes a major part of the French germplasm. We used molecular markers to characterise all accessions and to study genetic relationships between cultivars. Firstly, 497 olive trees were genotyped using 32 RAPD markers. We identified 114 RAPD profiles and detected several cases of mislabelling, synonymy and homonymy. Secondly, for each RAPD profile, one tree was analysed using mtDNA RFLPs to determine the cytoplasmic lineage of each cultivar and using five nuclear SSR loci. French germplasm displayed ME1, MOM and MCK mitotypes with ME1 prevailing (84%). Based on SSR markers, we revealed a slight differentiation between French cultivars growing in the West and the East side of the Rh?ne Valley. This study allowed us to construct a molecular data-base for the reference collection and to analyse genetic diversity for further prospecting, and for introducing new olive accessions.     

Utility of iPBS retrotransposons markers for molecular characterization of African Gnetum species

Abstract

Species of African Gnetum are lianas used as vegetables, medicines and for generating income. Despite the taxonomic confusion, identification of new species and diverse morphological characters in African Gnetum, molecular markers on these plants are lacking. However, the inter-primer binding site (iPBS) retrotransposons markers could be simple and excellent molecular markers for African Gnetum. The objective of this study was to determine the efficiency of iPBS markers in detecting genetic differentiation in African Gnetum species. A set of 21 iPBS markers were analysed on 14 accessions including G. africanum Welw., G. buchholzianum Engl. and the recently identified species G. latispicum. Six best selected primers generated 103 bands in G. africanum, 95 in G. buchholzianum and 24 in G. latispicum. Cluster analysis divided the accessions into two major groups. The first group contained all the accessions of G. africanum, whereas the second group was further divided in two subgroups representing accessions of G. buchholzianum and G. latispicum. Additionally, the Jaccard similarity coefficient indicated a close relationship between accessions of G. buchholzianum and G. latispicum. The iPBS marker system revealed genetic differentiation within African Gnetum and could be useful for evaluating genetic diversity, conservation, taxonomy and evolution studies.     

Identification of duplicates for the optimization of carrot collection management

Molecular markers have proved their efficiency for the identification of duplicate accessions in genetic resources collections. Partners of the GENRES Carrot project decided to evaluate the use of molecular markers for the identification of carrot accession duplicates. As a model analysis, 21 presumed duplicate accessions of Jaune du Doubs were selected. Only accessions that were not distinguished on a morphological basis were subjected to molecular analysis. The crucial question was to determine the threshold required to declare whether accessions were duplicates or not. We used a strategy based on the comparison between intravarietal and intervarietal genetic distances. DNA extractions were made on 4–8 bulks of five individuals per accession, and the bulks were analysed using 75 AFLP markers. An additional set of 7 bulks was extracted from one accession to provide true control replicates. With the exception of the true duplicates, all the accessions were clearly differentiated. Based on these results, a general strategy for the identification of carrot duplicates is proposed.     

番茄栽培品种SSR标记和形态标记的遗传多样性分析

比较了SSR标记和形态标记在研究番茄栽培品种遗传多样性上的应用。用7对SSR引物检测了11个番茄栽培品种的遗传多样性,共得到53条带,每一个位点上扩增出2-9条带,平均为6条;品种间遗传相似系数在0.39-0.84之间,平均遗传相似系数为0.60。根据11个形态学性状表型值计算的遗传相似系数在0.27-0.72之间,平均遗传相似系数0.58。用UPGMA进行聚类分析,SSR标记和形态标记都可依果肉颜色和果实大小将供试材料分组,即黄色和红色果肉,樱桃小番茄和大、中果形的混合型;两种方法对番茄栽培品种遗传多样性的评价相近。     

Morphological and molecular diversity reveal wide variability among sorghum Maldandi landraces from India

Maldandi is a popular sorghum variety for post-rainy or rabi cultivation in southern and central states of India, which is predominantly used for food purpose. Over time many landraces have been collected from these states which have vernacular connection with Maldandi. Genetic diversity among 82 Maldandi landraces, collected from such geographical regions was evaluated using both morphological (quantitative and qualitative) and SSR markers. In general, both morphological and SSR diversity revealed wide variability among the accessions studied. Euclidean distances based on 17 quantitative traits classified the accessions into two major clusters with two out groups, while the 19 qualitative traits clustered the accessions in one major cluster with six out groups. Sixteen out of 20 (80%) SSR markers detected polymorphism among the accessions with average PIC value of 0.36. Un-weighted neighbor joining clustering grouped the accessions into three clusters with 46, 16 and 17 accessions, respectively throwing three outliers. Average similarity coefficients of 0.62 and 0.34 based on morphological (qualitative) and SSR data indicated presence of wide variability among the Maldandi landraces. The standard check, M 35?C1 (a selection from the original Maldandi) could not be differentiated from EP 98, LG 2, LG 10, IS 4509 and IS 40791 based on qualitative data alone, while EP 54 and IS 33839 were indistinguishable from M 35?C1 solely using SSR markers. Either of the dendrogram threw unique grouping patterns with some identity. Thirteen promising Maldandi accessions selected based on field performance as well as morphological and molecular diversity could be used in the rabi improvement programme. SSR markers combined with morphological traits may effectively be used for designing breeding strategy and management of biodiversity and conservation of Maldandi genetic resources.     

Identification of the Worldwide Olive Germplasm Bank of Córdoba (Spain) using SSR and morphological markers

Olive is one of the most ancient crop plants and the World Olive Germplasm Bank of Cordoba (WOGBC), Spain, is one of the world’s largest collections of olive germplasm. We used 33 SSR (Simple Sequence Repeats) markers and 11 morphological characteristics of the endocarp to characterise, identify and authenticate 824 trees, representing 499 accessions from 21 countries of origin, from the WOGBC collection. The SSR markers exhibited high variability and information content. Of 332 cultivars identified in this study based on unique combinations of SSR genotypes and endocarp morphologies, 200 were authenticated by genotypic and morphological markers matches with authentic control samples. We found 130 SSR genotypes that we considered as molecular variants because they showed minimal molecular differences but the same morphological profile than 48 catalogued cultivars. We reported 15 previously described and 37 new cases of synonyms as well as 26 previously described and seven new cases of homonyms. We detected several errors in accession labelling, which may have occurred at any step during establishment of plants in the collection. Nested sets of 5, 10 and 17 SSRs were proposed to progressively and efficiently identify all of the genotypes studied here. The study provides a useful protocol for the characterisation, identification and authentication of any olive germplasm bank that has facilitated the establishment of a repository of true-to-type cultivars at the WOGBC.     

Genetic Diversity and Identity of Chinese Loquat Cultivars/Accessions (<Emphasis Type="Italic">Eriobotrya japonica</Emphasis>) Using Apple SSR Markers

Loquat (Eriobotrya japonica) is an underutilized fruit crop that originated in China and for which only a small number of molecular markers are available. This number can be increased by identifying apple SSRs that are transferable to loquat cultivars/accessions to provide new insight into the level of genetic diversity within loquat and synteny with apple. We evaluated 71 apple SSR markers distributed across 17 linkage groups, and identified 39 SSRs transferable to loquat. Testing 54 loquat accessions, from Japan, Spain, four provinces in China, and two wild species gave a total of 155 different alleles with a mean value of 3.38 per locus. The mean effective number of alleles was 2.21, and the mean observed heterozygosity was 0.47. These values indicate a high degree of genetic diversity in the set of Chinese loquat accessions analyzed. Unweighted pair-group method analysis based on simple matching coefficent clustered the accessions into two groups, cultivated and wild loquat. The cultivated loquat can be subdivided into three subgroups which generally reflect their geographic origin in China. The Spanish cultivars clustered with those of the Jiangsu and Zhejiang provinces. A core set of five SSR markers could distinguish most accessions.     

A consensus list of microsatellite markers for olive genotyping

Cultivar identification is a primary concern for olive growers, breeders, and scientists. This study was aimed at examining the SSR markers retrieved from the literature and currently used in olive study, in order to select those most effective in characterizing the olive accessions and to make possible the comparison of data obtained by different laboratories. Olive microsatellite profiles were assessed by four independent laboratories, which analyzed 37 pre-selected SSR loci on a set of 21 cultivars. These SSR markers were initially tested for their reproducibility, power of discrimination and number of amplified loci/alleles. Independent segregation was tested for each pair of SSRs in a controlled cross and the allelic error rate was quantified. Some of them were finally selected as the most informative and reliable. Most of the alleles were sequenced and their sizes were determined. Profiles of the reference cultivars and a list of alleles with their sizes obtained by sequencing are reported. Several genetic parameters have been analysed on a larger set of cultivars allowing for a deeper characterization of the selected loci. Results of this study provide a list of recommended markers and protocols for olive genotyping as well as the allelic profile of a set of reference cultivars that would be useful for the establishment of a universal database of olive accessions.     

Comparison of a retrotransposon-based marker with microsatellite markers for discriminating accessions of Vitis vinifera

Identification and knowledge concerning genetic diversity are fundamental for efficient management and use of grapevine germplasm. Recently, new types of molecular markers have been developed, such as retrotransposon-based markers. Because of their multilocus pattern, retrotransposon-based markers might be able to differentiate grapevine accessions with just one pair of primers. In order to evaluate the efficiency of this type of marker, we compared retrotransposon marker Tvv1 with seven microsatellite markers frequently used for genotyping of the genus Vitis (VVMD7, VVMD25, VVMD5, VVMD27, VVMD31, VVS2, and VZAG62). The reference population that we used consisted of 26 accessions of Vitis, including seven European varieties of Vitis vinifera, four North American varieties and hybrids of Vitis labrusca, and 15 rootstock hybrids obtained from crosses of several Vitis species. Individually, the Tvv1 and the group of seven SSR markers were capable of distinguishing all accessions except 'White Niagara' compared to 'Red Niagara'. Using the Structure software, the retrotransposon marker Tvv1 generated two clusters: one with V. vinifera plus North American varieties and the other comprising rootstocks. The seven SSR markers generated five clusters: V. vinifera, the North American varieties, and three groups of rootstock hybrids. The percentages of variation explained by the first two components in the principal coordinate analysis were 65.21 (Tvv1) and 50.42 (SSR markers) while the Mantel correlation between the distance matrixes generated by the two types of markers was 42.5%. We conclude that the Tvv1 marker is useful for DNA fingerprinting, but it lacks efficiency for discrimination of structured groups.     

Genetic analysis of Anatolian pear germplasm by simple sequence repeats

The pear (Pyrus communis L.) is a fruit species grown in many temperate regions of the world. Turkey harbours a rich and ancient pear germplasm adapted to diverse ecological regions of the country. The aim of this study was to genetically characterise locally grown Anatolian pear germplasm. We have analysed large numbers (228) of pear accessions originated from six eco‐geographically diverse regions using 18 simple sequence repeat (SSR) markers and identified 308 SSR alleles. Genetic similarities among the accessions examined were generally below 80%. The highest heterozygosity rate was obtained for the SSR locus ‘CH02D11’ derived from apples and ‘KA16’ and ‘NH0021a’ derived from pears. No identical or synonymous genotypes were found, while five homonymous genotypes were identified. Factorial correspondence analysis could not clearly separate different pear accession groups studied, suggesting that Anatolian pear accessions were intermixed possibly due to gene flow and/or germplasm movements between different eco‐geographical regions. However, most pear accessions were grouped according to their collection sites in structure analyses. The SSR data reported here for Anatolian pear accessions will be valuable for future germplasm management efforts as well as for comparative studies that investigate genetic relationships of pears from Anatolia and the surrounding regions.     

Genetic analysis of maize germplasm in the Korean Genebank and association with agronomic traits and simple sequence repeat markers

We assessed genetic and phenotypic variation in 105 maize germplasm accessions from RDA-Genebank of Korea and performed association analyses for 11 agronomical traits and 100 simple sequence repeats (SSR). Genetic diversity (GD) analysis revealed a total of 1104 alleles at the 100 SSR loci. The average number of alleles per locus was 11.0. The average GD and polymorphic information content values were 0.73 and 0.70, respectively. The average major allele frequency was 0.41. Population structure analysis indicated that these maize accessions comprised two major groups and one admixed group based on a membership probability threshold of 0.80. The two major groups contained 35 and 46 maize accessions. A mixed linear model of association analysis revealed five marker-trait associations with a significance level of P?≤?0.01 involving five SSR markers. A general linear model showed 72 marker-trait associations involving 42 SSR markers. We confirmed the presence in the general linear model associations of the five significant marker-trait associations (SMTAs) identified in the mixed linear model. For these SMTAs, two loci were associated with stem diameter and one locus each was associated with ear row number, leaf width, and leaf length. These results should prove useful for breeding new inbred lines by selecting parental lines using molecular markers and will help to preserve maize genetic resources in Korea.     

Construction of a SNP and SSR linkage map in autotetraploid blueberry using genotyping by sequencing

The construction of the first genetic map in autotetraploid blueberry has been made possible by the development of new SNP markers developed using genotyping by sequencing in a mapping population created from a cross between two key highbush blueberry cultivars, Draper × Jewel (Vaccinium corymbosum). The novel SNP markers were supplemented with existing SSR markers to enable the alignment of parental maps.  In total, 1794 single nucleotide polymorphic (SNP) markers and 233 simple sequence repeat (SSR) markers exhibited segregation patterns consistent with a random chromosomal segregation model for meiosis in an autotetraploid. Of these, 700 SNPs and 85 SSRs were utilized for construction of the ‘Draper’ genetic map, and 450 SNPs and 86 SSRs for the ‘Jewel’ map.  The ‘Draper’ map comprises 12  linkage groups (LG), associated with the haploid chromosome number for blueberry, and totals 1621 cM while the ‘Jewel’ map comprises 20 linkage groups totalling 1610 cM. Tentative alignments of the two parental maps have been made on the basis of shared SSR alleles and linkages to double-simplex markers segregating in both parents. Tentative alignments of the two parental maps have been made on the basis of shared SSR alleles and linkages to double-simplex markers segregating in both parents.     

葡萄品种DNA指纹数据库的构建及遗传多样性分析

利用SSR分子标记技术对314份葡萄品种进行了DNA指纹数据库构建和遗传多样性分析,为葡萄品种鉴定、亲缘关系分析和植物品种权保护提供科学依据。结果表明:9对引物共扩增出199个等位基因,多态性位点为199个,多态性比率达100%,每个标记检测到的位点数在17~31之间,平均为22.1个;多态性信息含量(PIC)值变幅在0.793~0.886之间,平均值为0.839。本研究发现3组同名异物品种和9组疑似同物异名品种,除此之外的290份品种中,70份品种仅需1对引物即可区分开,其余品种需要引物组合来实现品种之间的区分。最少选用8对引物即可完全区分开290份葡萄品种。最终利用8对多态性SSR引物构建了314份供试材料的DNA指纹数据库,聚类分析结果表明:263份二倍体供试材料可被分为真葡萄亚属和圆叶葡萄亚属两大类,而真葡萄亚属又被分为15个亚类。51份多倍体供试材料被分为3组,聚类结果与供试材料已知的系谱来源基本吻合。